1. Field of the Invention
The present invention relates generally to the fields of radiation and clinical oncology, radiotherapy, radioimmunobiology and nuclear medicine. More specifically, the present invention relates to a technique of targeting drug (or gene) carriers to select tissue via the up-regulation of adhesion molecules expressed on endothelial cells in response to exposure to radiation.
2. Description of the Related Art
Ionizing radiation (IR) is used widely to treat many conditions including cancer, arteriovenous malformations (AVM), macular degeneration, and intimal hyperplasia. Ionizing radiation therapy causes vascular lesions and damage in normal tissues. The microvasculature is quite sensitive to radiation (20) and is an important radiation dose-limiting factor in clinical applications. In almost all cases of therapeutic approach, the goal is to limit the exposure of normal tissue to the ionizing radiation while maximizing exposure to the diseased tissue. Indeed, improvement of techniques such as dose fractionation and conformal therapy (68), discovery of radioprotective drugs (78), and development of experimental methods of radiation therapy such as Microbeam Radiation Therapy (70) for reducing normal tissue toxicity of radiotherapy are currently active areas of research. In most cases, using modern clinical radiotherapeutic techniques, radiation damage can be limited to a core of diseased tissue and the immediate normal tissue surrounding it.
Ionizing radiation damage to the microcirculation is manifested in many forms including increased capillary permeability and up-regulation of inflammatory processes. An increase in permeability is an early and universal response of the microvasculature to ionizing radiation (19;46;50;51;75). For example, there is an increase in the blood-brain-barrier permeability in response to irradiation (22;62). Although this can lead to extravasation of blood proteins which may exacerbate tissue injury, the increased permeability can enhance delivery of chemotheraputic drugs across the blood-brain-barrier (61;62;64). Therefore, targeted drug delivery to irradiated tissue will not only provide a means to selectively deliver the drug but will also deliver the drug to a site of increased vascular permeability.
It has been known for over 15 years that exposure of normal and diseased tissue to irradiation causes an increase in leukocyte infiltration of the tissues (1;8;44;53;65;76). A key component of this process is the adhesion of leukocytes to the microvascular endothelium. A variety of studies focused on elucidating a detailed understanding of leukocyte adhesion in general (i.e. in response to stimuli other than radiation) have revealed that the movement of leukocytes from within the vasculature to the extravascular space involves a well orchestrated set of adhesion events (10;43;49;72). This adhesion cascade is mediated in part by adhesive bonds which form between glycoproteins (ligands) present on the leukocytes and cognate glycoproteins (receptors) present on the endothelium.
A key paradigm in this adhesion cascade is that certain endothelial cell adhesion molecules are inducible. That is, they are expressed at a low level, if at all, on endothelium within normal tissue, but dramatically up-regulated in response to appropriate biochemical stimuli (e.g. cytokines such as IL-1β) (10). Thus, in response to various cytokines, the endothelium becomes activated and increases its expression of receptors that bind ligands on the leukocytes. These receptors include E-selectin (CD62E), P-selectin (CD62P), VCAM-1 (CD54) and ICAM-1 (CD106).
Leukocytes attach to the endothelium, for the most part via the selectins, and begin to translate along the vessel wall (roll) at a velocity which is significantly lower than leukocytes in the free stream (72). As the leukocytes roll, they become activated in response to chemokines (18;72). The activation involves a number of changes to the leukocytes including an alteration in the density of the integrins on the leukocyte surface as well as an increase in the “stickiness” (a conformational change) of the integrins for their cognate endothelial cell adhesion molecules (e.g. ICAM-1) (15;56). The leukocytes firmly adhere to the endothelium via the integrins and proceed to migrate between adjacent endothelial cells into the extravascular space in part via PECAM-1 (CD31).
As noted above, a key component of leukocyte emigration is endothelial cell activation wherein the adhesion molecule profile on the lumenal surface of the endothelium is altered. Recognition of these drastically different endothelial surfaces has lead to the concept of endothelial cell adhesion molecule mediated targeted drug delivery (3;4;6;7;16;71). In this therapeutic approach, a drug would be incorporated into a carrier (e.g. a liposome (3;4;7;71) or a biodegradable particle (16;28)). The carrier would have a ligand for an endothelial cell adhesion molecule (e.g. E-selectin) that is selectively expressed on the target endothelial segment. Ideally the carrier would bind to the target endothelial segment (e.g. endothelium within a site of inflammation) via the selectively expressed receptor and not bind to non-target endothelium.
It is reasonable to anticipate that some of the molecular mechanisms involved in inflammatory processes initiated by insults other than radiation will also be operative in radiation induced inflammation. Recent literature suggests that this is, at least in part, true. In vitro studies aimed at characterizing the response of endothelial cells to irradiation have consistently shown ICAM-1 up-regulation on endothelial cells derived from large vessels (21;32;73) and vessels of the microvasculature (2;41). In vivo studies have also found up-regulation of ICAM-1 (12;35;36;42;47;53;58) and have ascribed increased leukocyte adhesion to the endothelium to an up-regulation of ICAM-1 (53;59). Indeed, radiation induced inflammatory response is significantly attenuated in mice deficient in ICAM-1 relative to wild type mice (35). In a recent clinical study (39) a significant increase in ICAM-1 expression in head and neck cancer patients treated with fractionated radiotherapy (30–60 Gy in 2 Gy daily fractions) has been reported.
At present the expression of E-selectin in response to radiation remains controversial. The expression of E-selectin has been studied in vitro using endothelial cells derived from large veins (i.e. HUVEC). One group reported significant up-regulation of E-selectin on human umbilical vein endothelial cells (31–33). In addition, this group found that the irradiated human umbilical vein endothelial cells supported E-selectin dependent adhesion of a leukocytic cell line (HL-60 cells) in semi-static adhesion assays (33). In contrast, others have found that E-selectin is not up-regulated on human umbilical vein endothelial cells in response to radiation (60; 21). It has also been found that irradiated human umbilical vein endothelial cells do not support the adhesion of HL-60 cells under in vitro flow conditions designed to mimic conditions present in vivo. Specifically, no adhesion of HL-60 cells were observed at shear stresses between 0.5–2.0 dynes/cm2 on post-IR human umbilical vein endothelial cells. Note that the lowest physiologically relevant in vitro shear stress is thought to be 0.5 dyne/cm2 (26). In contrast to the data on endothelial cells derived from large vein (i.e. human umbilical vein endothelial cells), a modest up-regulation of E-selectin on dermal microvascular endothelial cells (i.e. HDMEC) was observed which is in agreement with Heckman et al. (41). Consistent with this finding, in vitro flow adhesion assays revealed that post-IR dermal microvascular endothelial cells did support a small increase in HL-60 cell adhesion at relatively low (<=1.5 dynes/cm2) fluid shear. In vivo, it has been observed that there is an increase in the number of leukocytes which roll along the vessel wall in response to radiation (1;53;59). Consistent with this finding, E-selectin has been found within the microvasculature of the lung in response to radiation (36). A significant increase in E-selectin expression in head and neck cancer patients treated with fractionated radiotherapy (30–60 Gy in 2 Gy daily fractions) has also been reported (39).
A few studies have probed for the presence of VCAM-1 in response to radiation in vitro. VCAM-1 was observed to be up-regulated in irradiated skin microvascular endothelium (41) but not irradiated human umbilical vein endothelial cells (21;32). VCAM-1 was not up-regulated in head and neck cancer patients undergoing radiotherapy (39).
The expression of P-selectin post-IR has also been probed. One report found that P-selectin is localized to the vascular lumen of several irradiated tumors in vivo and increases in a time dependent manner until 24 hours post-IR (34). P-selectin is also reportedly translocated to the cell membrane in human umbilical vein endothelial cells within 30 minutes post-IR in vitro and in vivo. It is accumulated in the lumen of irradiated blood vessels in the lung and intestine but not in the brain or kidney (30;34;37).
Surface protein and mRNA levels of PECAM-1 (CD31), which is involved in the adhesion and transendothelial migration of leukocytes, has been shown to be up-regulated after irradiation in both human umbilical vein endothelial cells and tissue specimens from radiotherapy patients (63) but not in HDMEC (41). The up-regulation of PECAM-1 was found to be accompanied with increased transendothelial migration of leukocytes post-IR and this increased migration was inhibited with a mAb to PECAM-1 (63).
Although the issue of which endothelial cell adhesion molecules are expressed in response to radiation remains controversial, it is abundantly clear that the endothelial cell adhesion molecule profile is significantly altered in response to radiation. There is very convincing evidence that ICAM-1 and PECAM-1 are up-regulated. Although less clear, there is a modest amount of data suggesting that E-selectin is up-regulated as well. Even more noteworthy is that both ICAM-1 and E-selectin were significantly up-regulated in oral mucosa of head and neck cancer patients treated with radiotherapy (30–60 Gy in 2 Gy daily fractions) (39). The radiation induced up-regulation of endothelial cell adhesion molecules provides the opportunity to target drugs to select tissue via a combination of radiation and ligand-receptor drug targeting technology.
To clarify how the radiation therapy-targeted drug delivery scheme might work, consider the treatment of cancer as an example. Cancer patients are often treated with radiotherapy, chemotherapy or a combination of both. In an effort to limit side effects, the radiotherapy is designed to maximize radiation exposure to the cancerous tissue while minimizing exposure to normal tissue. Similarly, it would be ideal for a chemotherapeutic agent or a gene to be delivered only to the cancerous tissue and not to healthy tissue. Indeed, achieving this goal is the focus of a variety of drug delivery research.
In the combination radiation/targeting therapeutic model, a ligand-bearing drug carrier would be administered subsequent to, or in conjunction with, the radiotherapy. A variety of materials could be used for the drug carrier including liposomes or carriers made from biodegradable polymers. The drug carrier would contain a therapeutic agent (e.g. an organic compound, or a nucleic acid) and, on its outer surface, a recognition molecule (ligand) for a cognate molecule (receptor) that is expressed selectively (due to exposure to the radiation) on the lumenal surface of the endothelium within the irradiated tissue. Ideally, these carriers would bind predominately within the vasculature of the irradiated tissue (i.e. the cancerous tissue) and not bind to the vasculature of normal tissue. In this manner, the radiation induced up-regulation of a endothelial cell adhesion molecule(s) within the diseased tissue is used as a target to deliver therapeutic agents (drugs, genes, etc.) selectively to the site of disease.
The prior art is deficient in the ability to target drug (or gene) carriers to select tissue via the up-regulation of adhesion molecules expressed on endothelial cells in response to exposure to radiation. The present invention fulfills this long-standing need and desire in the art.